Amplification of DNA by polymerase chain reaction (PCR) is a technique fundamental to molecular biology. Nucleic acid analysis by PCR requires sample preparation, amplification, and product analysis. Although these steps are usually performed sequentially, amplification and analysis can occur simultaneously. DNA dyes or fluorescent probes can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination for subsequent reactions, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real time” PCR. See, for example, U.S. Pat. No. 6,174,670.
It is known for a long time that ionic concentration strongly affects the melting temperature of double stranded DNA hybrids. It is also known for a long time that metal ions or other ions are capable of binding to or interacting with nucleic acids and nucleic acid polymerases such as the thermostable Taq polymerase used for PCR. As a consequence, adding of such specific ions in many cases has been proven to have a strong impact on the performance of an amplification reaction. In particular, it is known that depending on the actual primer and target nucleic acid sequences, each different PCR reaction has a specific and very narrow optimum for its magnesium ion concentration.
A major problem with nucleic acid amplification and more especially with PCR is the generation of nonspecific amplification products such as the formation of primer/dimer artifacts. In many cases, this is due to a nonspecific oligonucleotide priming and subsequent primer extension event prior to the actual thermocycling procedure itself, since thermostable DNA polymerases are also moderately active at ambient temperature. For example, amplification products due to by chance occurring primer dimerization and subsequent extension are observed frequently.
Extension of non-specifically annealed primers has been shown to be inhibited by the addition of short double stranded DNA fragments (Kainz, P., et al., Biotechniques 28 (2000) 278-282). In this case, primer extension is inhibited at temperatures below the melting point of the short double stranded DNA fragment, but independent from the sequence of the competitor DNA itself. However, it is not known, to which extent the excess of competitor DNA influences the yield of the nucleic acid amplification reaction.
Alternatively, oligonucleotide aptamers with a specific sequence resulting in a defined secondary structure may be used. Such aptamers have been selected using the SELEX Technology for a very high affinity to the DNA polymerase (U.S. Pat. No. 5,693,502; Lin, Y., and Jayasena, S. D., J. Mol. Biol. 271 (1997) 100-111). The presence of such aptamers within the amplification mixture prior to the actual thermocycling process itself again results in a high affinity binding to the DNA polymerase and consequently a heat labile inhibition of its activity.
Due to the selection process, however, all so far available aptamers can only be used in combination with one particular species of DNA polymerase.
Oligonucleotide inhibitors (WO 01/02559) with blocked 3′ end and 5′ end were shown to enhance sensitivity and specificity of PCR reactions. They compete with the primer in binding to the polymerase.
It is also known that addition of single strand binding protein (U.S. Pat. No. 5,449,603) or tRNA (Sturzenbaum, S. R., Biotechniques 27 (1999) 50-52) results in non-covalent association of these additives to the primers. This association is disrupted when heating during PCR. It was also found that addition of DNA helicases prevent random annealing of primers (Kaboev, O. K., et al., Bioorg Khim 25 (1999) 398400). Furthermore, poly-glutamate (WO 00/68411) in several cases may be used in order to inhibit polymerase activity at low temperatures.
Other organic additives known in the art like DMSO, betaines, and formamides (WO 99/46400; Hengen, P. N., Trends Biochem. Sci. 22 (1997) 225-226; Chakrabarti, R., and Schutt, C. E., Nucleic Acids Res. 29 (2001) 2377-2381) result in an improvement of amplification of GC rich sequences, rather than prevention of unspecific priming. Similarly, heparin may stimulate in vitro run-on transcription presumably by removal of proteins like histones in order to make chromosomal DNA accessible (Hildebrand, C. E., et al., Biochimica et Biophysica Acta 477 (1977) 295-311). In particular, WO 99/46400 discloses the beneficial effects on PCR performance of adding compounds such as 4-methylmorpholine N-oxide, betaine, and N-alkylimidazoles such as proline, 1-methylimidazole or 4-methylimidazole.
Yet, unfortunately, none of the PCR additives disclosed in the art so far is capable of providing an absolute specificity for primer annealing while maintaining sufficient sensitivity.
Thus, there is a need in the art to provide an alternative PCR method, which is cheap, easy to perform, and—most important—which does improve the performance of the PCR reaction itself.